Pseudomonas aeruginosa persists as a major cause of life-threatening infections for individuals with the following conditions: cystic fibrosis, burns, wounds, cancer (leukemias), those receiving immunosuppressive therapy, diabetics, as well as intravenous drug users. The pili of this bacterium are protein filaments that extend from the ends of the cell. These structures serve as adhesion factors, and so are important virulence factors. The long-term objectives of this project are to determine the role of pilus glycosylation in P. aeruginosa pathogenesis and to ascertain the importance of the glycan in pilus vaccine design. Work from my laboratory has shown that the pili of P. aeruginosa 1244 are glycosylated with a trisaccharide moiety originating in the lipopolysaccharide O-antigen biosynthetic pathway. The research in this proposal aims to determine the structural relationship between the pilin glycan and the O-antigen. In addition, it will explore the ability of the glycosylation system to attach heterologous glycan to pilin. The project will determine the glycan precursor involved in pilin glycosylation and elucidate the point in pilus biogenesis where glycosylation occurs. The project will determine the step in O-antigen biosynthesis that produces the glycan and the subcellular location of pilin glycosylation.